The recently described RNA interference (RNAi) phenomenon mediated by small interfering RNAs (siRNAs) is highly sequence specific gene silencing mechanism. Recent studies established the remarkable potency of siRNAs in suppressing HIV-1 replication in vitro and highlighted its potential as 3owerful tool for gene therapy. Translation of this promising new technology into the clinic requires stable introduction of the siRNA genes into hematopoietic stem cells and derivation of viral resistant T cells and macrophages. Using in vitro assays and a unique SCID-hu mouse in vivo model that harbors transplanted human tissue, we recently showed that CD34 hematopoietic progenitor cells transduced with anti-HIV siRNAs, ribozymes and RNA decoys could be differentiated into virus resistant T cells and macrophages. These proofs of concept studies paved the way to evaluate novel approaches in an in vivo setting. Several new developments have also occurred recently in the areas of stem cell biology, lentiviral vectors, and in vivo modeling with direct relevance to HIV gene therapy. In the current proposal our goal is to build upon our recent progress. Our specific objectives are: 1. Evaluate the synergistic efficacy of novel combinatorial siRNA constructs targeted to different stages of HIV-1 life cycle in vitro and analyze their mechanism of action. 2. Transduce siRNAs via lentiviral vectors into CD34 hematopoietic progenitor cells to derive HIV-1 resistant macrophages and investigate the mechanism of their action in differentiated cells. 3. Determine the in vivo protective effects of different anti-HIV-1 siRNAs, either individually or in combination, in SCID-hu mice thy/liv grafts against HIV-1 challenge and also assess the functional competence of transgenic T cells. 4. Transduce siRNA constructs into the newly described CD34+/KDR+ primitive hematopoietic progenitor cells and derive macrophages in vitro and thymocytes in vivo, and evaluate their HIV-1 resistance. [unreadable] [unreadable]